Compositions comprising molecules that bind PCSK9 and methods of use

Abstract

The present disclosure provides pharmaceutical compositions comprising fibronectin-based scaffold domain proteins that bind, for example, proprotein convertase subtilisin-kexin-9 (PCSK9).

Description

[0001] Related Applications

[0002] This application claims the benefit of and priority to U.S. Provisional Application No. 62 / 672,187, filed May 16, 2018, which is incorporated herein by reference in its entirety. Technical Field

[0003] The present disclosure relates to fibronectin-based scaffold domain proteins that bind proprotein convertase subtilisin kexin-9 (PCSK9), as well as pharmaceutical compositions and methods of use thereof. Background Art

[0004] Proprotein convertase subtilisin / kexin type 9 (PCSK9) is an enzyme encoded by the PCSK9 gene on human chromosome 1. PCSK9 binds to receptors for low-density lipoprotein particles (LDL). LDL receptors (LDLRs) on the membranes of liver and other cells bind and initiate endocytosis of LDL particles from the extracellular fluid into cells, thereby reducing the concentration of circulating LDL particles. If PCSK9 is blocked, more LDLRs are recycled and present on the cell surface, thereby removing LDL particles from the extracellular fluid. Therefore, blocking PCSK9 can reduce the concentration of blood LDL particles.

[0005] The monoclonal antibody PCSK9 inhibitors alirocumab and evolocumab are approved for biweekly or monthly subcutaneous injection or infusion to reduce LDL particle concentrations when statins and other drugs are ineffective or poorly tolerated. For monthly injections, several milliliters of drug product are required to achieve the desired dose. Although formulations with higher concentrations of active agent can provide more ideal dosing schedules and volumes, these formulations are hampered by solubility limitations, increased viscosity, and instability of biologics, including the tendency to aggregate and form particulates. Carpenter JF et al., Qverlooking subvisible particles in therapeutic protein products:Gaps that may compromise product q u ality , Journal of Pharmaceutical Sciences, Vol. 98, No. 4 (2008).

[0006] There is a need for pharmaceutical compositions that target PCSK9 and have the potential to achieve more optimal dosing schedules, smaller volumes, and / or improvements in efficacy while maintaining an overall safe and well-tolerated profile. Summary of the invention

[0007] The present disclosure provides a composition comprising a fibronectin scaffold protein that binds to proprotein convertase subtilisin kexin type 9 (PCSK9) with high affinity and can be stably formulated at high concentrations to achieve maximum biological effect and more convenient dosing schedules, dosing volumes, and patient-friendly delivery devices. Fusion proteins that bind to PCSK9 include a motif that binds to PCSK9 and an amino acid sequence of human serum albumin (HSA). The motif that binds to PCSK9 and the HSA amino acid sequence can be expressed as a gene fusion or chemically conjugated.

[0008] The motifs described herein that bind to PCSK9 are based on adnectin, a family of proteins derived from the human 10th fibronectin type III domain (10Fn3) that are engineered to achieve high affinity target binding. According to the present disclosure, fusion proteins that bind to PCSK9 are stably formulated at high concentrations to achieve maximum biological activity and convenient dosing schedules and volumes. The concentration of the fusion protein that binds to PCSK9 in the composition is at least 100 mg / mL. In some embodiments, the concentration of the fusion protein that binds to PCSK9 in the composition is at least about 200 mg / mL. In some embodiments, the concentration of the fusion protein that binds to PCSK9 in the composition is at least about 250 mg / mL, or at least about 275 mg / mL, or at least about 300 mg / mL, or at least about 350 mg / L. In some embodiments, the fusion protein that binds to PCSK9 is administered at a unit dose of about 275 mg to about 325 mg (e.g., about 300 mg).

[0009] As described herein, the PCSK9-binding motif comprises or consists of the amino acid sequence of (SEQ ID NO: 1) or a variant thereof. The PCSK9-binding motif binds to human PCSK9 with subnanomolar affinity in a concentration-dependent manner. The PCSK9-binding motif is chemically conjugated or C-terminally fused to a human serum albumin (HSA) amino acid sequence. In various embodiments, the PCSK9-binding motif is fused to the HSA amino acid sequence at the C-terminus and may include a linker sequence of amino acids between the PCSK9-binding motif and the HSA amino acid sequence.

[0010] Fusion proteins that bind PCSK9 can be stably formulated in solution form at high concentrations. The formulations do not show significant degradation or particle formation under conventional long-term storage conditions and short-term storage under accelerated and pressurized conditions. (In addition to the active agent) exemplary formulations include or are essentially composed of: L-histidine, L-histidine monohydrochloride, sodium chloride, and optional polysorbate 80.

[0011] The pharmaceutical composition of the present disclosure can conveniently exist in a unit dosage form, and the unit dosage form contains a predetermined amount of the disclosed activating agent per dose. In some embodiments, the volume of the unit dose does not exceed about 1.5mL, or the volume does not exceed about 1mL. In some embodiments, the volume of the unit dose does not exceed 0.8mL, or the volume does not exceed 0.7mL. In other embodiments, with a micro dose, for example, with a volume less than 0.5mL, or less than about 0.25mL, or less than about 0.15mL, the composition is applied. In various embodiments, the composition is delivered with a unit dose of a fusion protein comprising about 20 to about 450mg of PCSK9. For example, in some embodiments, a dosage of 20 to about 75mg is applied with a weekly micro dose (for example, with a volume less than about 0.25mL or less than about 0.15mL). In other embodiments, a dosage of about 200 to about 450mg is applied with a volume in the range of about 0.7 to 1.5mL about every two weeks, every month, or every other month.

[0012] The composition or preparation is suitable for administration by subcutaneous, intramuscular, intradermal or intravenous administration. The high concentration preparation allows a less frequent dosing schedule and a lower dosing volume to be suitable for subcutaneous administration. As demonstrated herein, the maximum PCSK9 inhibition is achieved under a relatively low concentration of the fusion protein in conjunction with PCSK9, and a higher concentration achieves a longer inhibition duration. In some embodiments, the subject receives a unit dose of the composition approximately once a week, once every 2 weeks, or approximately once every 3 weeks, or approximately once every 4 weeks (e.g., approximately once a month), or approximately once every 6 weeks, or approximately once every 8 weeks (approximately once every 2 months).

[0013] In some embodiments, the subject receives a microdose of the composition about weekly or about biweekly, such as in a volume of about 50 to about 250 μL.

[0014] The composition can be used to treat PCSK9 related disorders in human subjects. In some embodiments, the patient needs to reduce LDL (e.g., LDL-C). In some embodiments, the subject may show cholesterol related diseases, such as hypercholesterolemia and / or atherosclerosis. In some embodiments, the subject suffers from familial hypercholesterolemia. In some embodiments, the subject suffers from cardiovascular disease (e.g., atherosclerotic coronary heart disease) or is at high risk of cardiovascular disease.

[0015] In some embodiments, the composition is administered with statin therapy or another oral lipid-lowering therapy, or in some embodiments, the composition is provided as the sole therapy for hypercholesterolemia, i.e., without oral lipid-lowering therapy (e.g., statin therapy). BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The structure of the fusion protein binding to PCSK9 is shown.

[0017] Figure 2 PCSK9 pharmacokinetic and pharmacodynamic (PK-PD) models in non-human primates (NHPs) and humans are shown. LIB-003 refers to a fusion protein that binds PCSK9.

[0018] Figure 3 Gadkar model for predicting the effect of PCSK9 targeting on LDLC is shown. Gadkar K et al., A Mechanistic Systems Pharmacology Model for Prediction of LDL Cholesterol Lowering by PCSK9 Antagonism Human Dyslipidemic Populations ,CPTPharmacometrics Syst.Pharmacol.2014;3(11). DETAILED DESCRIPTION

[0019] The present disclosure provides a composition comprising a fibronectin scaffold fusion protein, which binds to proprotein convertase subtilisin kexin-9 (PCSK9) with high affinity and can be stably formulated at high concentrations to achieve maximum biological effect and more convenient dosing schedule and dosing volume, and delivered using a patient-friendly delivery device (such as a syringe or an automatic injector). The fusion protein that binds to PCSK9 comprises a motif that binds to PCSK9 and an amino acid sequence encoding human serum albumin (HSA) at the C-terminus.

[0020] Proprotein convertase subtilisin / kexin type 9 is a circulating protein secreted primarily by the liver that plays an important role in the recycling of hepatic LDLR and has been identified as an effective drug target for lowering LDL-C. LDLR is the major pathway for the clearance of LDL-cholesterol (LDL-C) from the circulation. Plasma PCSK9 binds to hepatic LDLR along with LDL-C, which targets the receptor for endocytosis and degradation, thereby reducing the effectiveness of LDLR in clearing LDL-C from the circulation. By inhibiting the binding of PCSK9 to LDLR, LDLR degradation can be prevented, LDLR recycling can be increased, LDL-C clearance can be improved, and circulating LDL-C levels can be reduced.

[0021] The PCSK9 binding motifs described herein are based on "adnectins," a family of proteins derived from the human 10th fibronectin type III domain that are engineered to achieve high affinity target binding. "Adnectins" are small (≤12 kDa), compact proteins that have no sequence homology to immunoglobulins but have a β-sheet structure with diversified loops similar to antibody variable regions. Adnectins do not have disulfide bonds and are not glycosylated, exhibit high thermal stability and monomeric solution behavior, and can be efficiently produced using bacterial, yeast, or mammalian expression systems. By modifying the variable loop sequences and lengths while keeping the scaffold residues essentially constant, subnanomolar target binding affinities can be achieved while maintaining structural stability. Given their size, adnectins are rapidly filtered by the kidneys, and therefore require pharmacokinetic (PK) enhancement modifications to achieve in vivo applications. Exemplary PCSK9 binding motifs (adnectins) are disclosed in U.S. Pat. Nos. 8,420,098; 9,234,027; and 9,856,309, each of which is incorporated herein by reference in its entirety.

[0022] According to the present disclosure, the fusion protein that binds PCSK9 is stably formulated at high concentrations to achieve maximum biological activity and convenient dosing schedules and volumes. The concentration of the fusion protein that binds PCSK9 in the composition is at least 100 mg / mL. In some embodiments, the concentration of the fusion protein that binds PCSK9 in the composition is at least about 150 mg / mL, or in some embodiments, at least about 175 mg / mL, or at least about 200 mg / mL, or at least about 225 mg / mL. In some embodiments, the concentration of the fusion protein that binds PCSK9 in the composition is at least about 250 mg / mL, or at least about 275 mg / mL, or at least about 300 mg / mL, or at least about 350 mg / mL. In some embodiments, the concentration of the fusion protein that binds PCSK9 in the composition is about 250 mg / mL to about 350 mg / mL. In some embodiments, the concentration of the fusion protein that binds PCSK9 in the composition is about 250 mg / mL or about 300 mg / mL.

[0023] The PCSK9 binding motif comprises or consists of the amino acid sequence of (SEQ ID NO: 1):

[0024] VSDVPRDLEVVAATPTSLLISW DAPAEGYG YYRITYGETGGNSPVQEFTV PVSKG TATISGLKPGVDYTITVYAV EFDFPGAGYYHR PISINYRTE.

[0025] The loops that bind PCSK9 are underlined. In some embodiments, the motif that binds PCSK9 is a variant of SEQ ID NO: 1 having one to five amino acid substitutions, deletions, or insertions relative to SEQ ID NO: 1. In some embodiments, the amino acid changes are made outside the binding loop. In some embodiments, 1, 2, or 3 amino acid changes are made within the binding loop.

[0026] The PCSK9-binding motif is designed to specifically target PCSK9 while essentially maintaining wild-type (WT) 10 Fn3 sequences to minimize inherent immunogenicity. See U.S. Patent No. 8,420,098, which is incorporated herein by reference in its entirety. The PCSK9-binding motif binds to human PCSK9 with subnanomolar affinity in a concentration-dependent manner.

[0027] In various embodiments, the motif that binds PCSK9 is fused to the human serum albumin (HSA) amino acid sequence at the C-terminus. In some embodiments, HSA comprises a sequence having at least 80% identity, or at least 85% identity, or at least 90% identity, or at least 95% identity, or at least 98% identity, or at least 99% identity to the amino acid sequence of SEQ ID NO: 2. For example, the HSA amino acid sequence may comprise one to ten or one to five modifications independently selected from amino acid substitutions, deletions, and insertions relative to SEQ ID NO: 2. In some embodiments, the HSA amino acid sequence comprises an alanine residue at a position corresponding to position 34 of SEQ ID NO: 2, as shown in SEQ ID NO: 2. In various embodiments, the length of the HSA amino acid sequence is at least 500 amino acids.

[0028] In some embodiments, the PCSK9 binding motif and the HSA amino acid sequence are chemically conjugated using any known chemical conjugation method.

[0029] HSA is a multidomain protein composed of a cluster of helices, containing 17 pairs of disulfide bridges; in native HSA, only one cysteine ​​residue, Cys34, exists as a free thiol group. In some embodiments, this Cys is replaced by an alanine residue in a fusion protein that binds to PCSK9. The HSA portion is used to extend the circulating half-life of the fusion protein that binds to PCSK9. In various embodiments, the fusion protein that binds to PCSK9, comprising a motif that binds to PCSK9 and an HSA amino acid sequence, has a molecular weight of about 77 kDa.

[0030] In some embodiments, the HSA amino acid sequence is a variant described in U.S. Patent 9,493,545, U.S. Patent 9,821,039, U.S. Patent 9,944,691, or U.S. 2014 / 0315817, each of which is herein incorporated by reference in its entirety.

[0031] In some embodiments, the motif that binds PCSK9 and the HSA amino acid sequence are connected by gene fusion, for example, to the HSA amino acid sequence at the C-terminus of the molecule. A short amino acid linker can connect the domain that binds PCSK9 and the HSA amino acid sequence. For example, the linker can comprise 2 to 20 amino acids, or in some embodiments, 4 to 10 amino acids. In some embodiments, the motif that binds PCSK9 and the HSA amino acid sequence are connected via a 6-amino acid linker. The linker can be composed primarily of serine, glycine, threonine, and alanine amino acids. For example, the linker can be a serine / glycine linker. In some embodiments, the linker comprises or consists of the amino acid sequence GSGSGS.

[0032] Fusion proteins that bind to PCSK9 can be stably formulated in solution form at high concentrations. For example, under long-term storage conditions, such as a temperature of about 5°C (e.g., 2°C to 8°C) or under short-term storage conditions under ambient conditions (e.g., 1 to 6 months at 25±3°C), the formulation does not show significant degradation or particle formation. Fusion proteins that bind to PCSK9 are suspended in an appropriate physiological solution (e.g., saline or other pharmacologically acceptable solvents or buffer solutions) and may optionally contain a surfactant (e.g., a nonionic surfactant). In some embodiments, the formulation comprises a buffer, an isotonic agent, an optional surfactant, and a solvent.

[0033] Pharmaceutically acceptable carriers include water, saline, glycerol. In some embodiments, the formulation may include fixed oils, polyethylene glycol, propylene glycol or other solvents. In some embodiments, the solvent is water.

[0034] The preparation is usually a buffer solution. As used herein, "buffer" refers to a chemical agent that can absorb a certain amount of acid or alkali without experiencing a strong change in pH. Exemplary buffers include citrate buffer, phosphate buffer, acetate buffer, succinate buffer and bicarbonate buffer. In some embodiments, the buffer may include L-histidine / L-histidine monohydrochloride. For example, when using L-histidine / L-histidine monohydrochloride buffer system, L-histidine / L-histidine monohydrochloride can be about 1mg / mL to about 10mg / mL, such as about 2mg / mL to about 5mg / mL. In some embodiments, the pH of the preparation is maintained in the range of about pH 5.5 to about pH 7.2 by the buffer, such as about pH 6.8 (for example, pH 6.6 to 7.0). In some embodiments, the pH of the preparation is adjusted, for example, with hydrochloric acid and / or sodium hydroxide. In some embodiments, the ratio of L-histidine / L-histidine monohydrochloride is such that pH adjustment is not required.

[0035] In various embodiments, isotonic agents that can be used alone or in combination include dextrose, sucrose, glycerol, trehalose, mannitol, sorbitol, arginine, sodium chloride or potassium chloride. In some embodiments, isotonic agents include sodium chloride or consist of sodium chloride. For example, the formulation may include about 2 to about 20 mg / mL of sodium chloride (e.g., 6 to 12 mg / mL sodium chloride), or one or more isotonic agents equal to the amount of 2 to 20 mg / mL sodium chloride (or 6 to 12 mg / mL sodium chloride) weight gram molecular osmotic concentration.

[0036] In some embodiments, the formulation comprises a surfactant, which can act as a solubilizing agent. In some embodiments, the surfactant is a nonionic surfactant. Exemplary nonionic surfactants include polysorbate surfactants, such as polysorbate 20, 40, 60, or 80. For example, the formulation may include polysorbate 80. Other pharmaceutically acceptable nonionic surfactants may also be used alone or in combination. In some embodiments, the formulation does not include a surfactant.

[0037] Exemplary formulations include citrate buffer (e.g., 10-50 mM, pH 5.6 to 6.0), histidine buffer (e.g., 10 mM to 50 mM, pH 6.0 to 7.0) or succinate buffer (e.g., 10 to 50 mM, pH 5.5 to 6.0). In some embodiments, the formulation comprises an excipient selected from arginine (e.g., 100 to 200 mM), NaCl (e.g., 100 to 200 mM), sorbitol (e.g., 100 to 300 mM) or sucrose (e.g., 100 to 300 mM). In some embodiments, the formulation comprises a surfactant, such as polysorbate 80 (e.g., 0.01 to 0.5 mg / mL). In some embodiments, the formulation does not contain a surfactant.

[0038] In some embodiments, the formulation further comprises a preservative, such as phenol, m-cresol, or sodium benzoate.

[0039] In some embodiments, the fusion protein formulation that binds PCSK9 consists essentially of L-histidine, L-histidine monohydrochloride, sodium chloride, and polysorbate 80 (in addition to the active agent). Table 1 Display:

[0040]

[0041] The pharmaceutical compositions of the disclosure may conveniently be presented in unit dosage form containing a predetermined amount of the active agent of the disclosure per dosage.

[0042] In some embodiments, the composition is contained in an injection pen. An automatic injector such as an "injection pen" is a spring-loaded syringe designed to deliver a certain dose of a specific drug. By design, the injection pen is easy to use and is intended to be administered by the patient himself or by untrained personnel. The injection pen is designed to overcome the hesitation associated with the self-administration of needle-based drug delivery devices. The injection pen keeps the needle tip hidden before injection and has a passive safety mechanism to prevent accidental firing (injection). The injection depth can be adjusted or fixed, and the function of removing the needle shield can be combined. By pressing a button, the syringe needle will automatically insert into the subcutaneous tissue and deliver the drug. After the injection is completed, some injection pens will have visual or auditory indications to confirm that the full dose has been delivered.

[0043] In some embodiments, the injection pen contains 1 to 10 unit doses or 1 to 5 unit doses. In some embodiments, the volume of the unit dose does not exceed about 1.5mL or about 1mL (whether or not contained or delivered by the injection pen). In some embodiments, the volume of the unit dose does not exceed 0.8mL, or the volume does not exceed 0.7mL. In some embodiments, the injection pen delivers a microdose, for example, with a volume in the range of about 50 μL to about 500 μL, or a volume in the range of about 75 μL to about 250 μL. In some embodiments, the microdose has a volume of 100 to 200 μL. In various embodiments, the injection pen or other devices for subcutaneous delivery provide a dose of a fusion protein with 30 to about 450mg of PCSK9 binding. In various embodiments, the unit dose is about 50 to about 400mg, or about 50 to about 300mg. In some embodiments, the unit dose is at least 200mg, or at least 250mg, or at least 300mg. In some embodiments, the unit dose is about 250 mg to about 350 mg (e.g., about 300 mg). The amount of the active agent delivered per unit dose can be adjusted based on the desired frequency of administration. For example, in some embodiments, a dose of 20 to about 75 mg is administered with a weekly microdose (e.g., with a volume less than about 0.25 mL or less than about 0.15 mL). In other embodiments, a dose of about 200 to about 450 mg is administered with a volume in the range of about 0.7 to 1.5 mL approximately every two weeks, monthly, or every other month. In some embodiments, a dose of about 275 mg to about 350 mg (e.g., about 300 mg) is administered by subcutaneous injection every four weeks with a volume of about 1.5 mL or less or about 1 mL or less.

[0044] The composition or preparation is suitable for administration by subcutaneous, intramuscular, intradermal or intravenous administration. The high concentration, relatively low viscosity and appropriate weight-gram molecular osmotic concentration of the preparation realize patient tolerance, less frequent administration schedule and smaller volume. As demonstrated herein, the maximum PCSK9 inhibition is achieved under the fusion protein of relatively low concentration in conjunction with PCSK9, and higher concentration realizes longer inhibition duration. In some embodiments, the subject receives a unit dose of the composition about once every 1 week (e.g., by applying a microdose once a week), or about once every 2 weeks, or about once every 3 weeks, or about once every 4 weeks (e.g., about once a month), or about once every 6 weeks, or about once every 8 weeks (about once every 2 months).

[0045] The composition can be used to treat PCSK9 related disorders in human subjects. PCSK9 related disorders are described in U.S. Patents 8,420,098, 9,238,027 and 9,856,306, which are incorporated herein by reference in their entirety. In some embodiments, the patient needs to reduce LDL (e.g., LDL-cholesterol). In some embodiments, the subject may show cholesterol related diseases, such as hypercholesterolemia and / or atherosclerosis. In various embodiments, the subject shows a disease selected from lipid disorders, hypercholesterolemia, hyperlipoproteinemia, hyperlipidemia, dyslipidemia, coronary heart disease, atherosclerosis and diabetes. In some embodiments, the subject suffers from familial hypercholesterolemia. In some embodiments, the subject suffers from cardiovascular disease (e.g., atherosclerotic coronary heart disease) or is at high risk of cardiovascular disease.

[0046] Hypercholesterolemia is a disease characterized by elevated serum cholesterol. Elevated serum cholesterol levels affect a significant portion of the population and are an important risk factor for atherosclerosis and myocardial infarction. Cholesterol-lowering drugs such as HMG-CoA reductase inhibitors ("statins") are routinely administered to hypercholesterolemia patients. "Familial hypercholesterolemia" (FH) is a hereditary disorder characterized by high cholesterol levels in the blood and early cardiovascular disease, particularly very high low-density lipoprotein (e.g., LDL-cholesterol) levels. For individuals suffering from FH, high cholesterol levels are less responsive to conventional cholesterol control methods (such as statin therapy).

[0047] FH is an autosomal dominant inherited metabolic disorder characterized by one or more mutations in the LDL receptor (LDL-R) gene or other genes involved in lipid regulation, significantly elevated LDL-C and early onset of atherosclerosis. In some embodiments, hypercholesterolemia is homozygous familial hypercholesterolemia or HoFH, which is a condition characterized by mutations in both the maternal and paternal LDL-R genes.

[0048] In some embodiments, the subject has heterozygous FH. Heterozygous FH is typically treated with statins, bile acid sequestrants, or other lipid-lowering agents that lower cholesterol levels.

[0049] In some embodiments, hypercholesterolemia is polygenic hypercholesterolemia, which is a condition characterized by elevated cholesterol and is caused by the influence of multiple genetic factors. In certain embodiments, polygenic hypercholesterolemia can be exacerbated by dietary lipid intake.

[0050] In some embodiments, the composition is administered together with statin therapy or other oral lipid-lowering therapies. In such embodiments, the composition will provide a cumulative reduction in LDL-C. In some embodiments, the composition is provided as the only therapy for hypercholesterolemia, i.e., without statin therapy or other oral lipid-lowering therapies. For example, in such embodiments, the subject may have statin intolerance. "Statin intolerance" occurs when a patient is unable to continue using statins due to side effects or abnormalities indicating liver function or muscle function (creatine kinase) after a blood test. In some embodiments, statin intolerance can be partial (i.e., only certain doses of certain statins) or complete (i.e., all statins at any dose). In some embodiments, statin intolerance causes muscle aches, pains, weakness, or cramps (i.e., myalgia); occurs in up to 15% of patients treated.

[0051] Unless otherwise stated, the term "about" as used herein means ± 10% of the associated numerical value.

[0052] Embodiments of the present invention will now be described by way of the following examples.

[0053] Example

[0054] Example 1: PCSK9-binding fusion protein and PK-PD behavior

[0055] The PCSK9-binding fusion protein comprises a modified fibronectin domain targeting proprotein convertase subtilisin / Kexin type 9 (PCSK9) and human serum albumin ( Figure 1 ). The total molecular weight of the fusion protein is approximately 77,000 Daltons. The PCSK9-binding fusion protein has a high binding affinity for human PCSK9 and a >100-fold weaker affinity for cynomolgus monkey PCSK9. Despite this difference in binding affinity, non-human primates (NHPs) are considered an appropriate species for testing the safety and pharmacology of PCSK9-binding fusion proteins because the greatest degree of free PCSK9 inhibition and the greatest degree of LDL-C reduction are achieved in NHPs.

[0056] Fusion proteins that bind to PCSK9 are being developed for subcutaneous (SC) administration to treat, for example, hypercholesterolemia, including patients with familial hypercholesterolemia or hypercholesterolemia and atherosclerotic coronary heart disease (CHD). The two main determinants of the PK of fusion proteins that bind to PCSK9 are interaction with its target PCSK9, and the ability to recycle via the neonatal Fc receptor (FcRn) and minimize renal filtration, thereby reducing the clearance of the fusion protein that binds to PCSK9 and increasing the half-life. Studies conducted in wild-type mice cannot address the impact of these determinants on the PK of fusion proteins that bind to PCSK9, because fusion proteins that bind to PCSK9 do not effectively bind to mouse PCSK9, and HSA does not interact with mouse FcRn. Therefore, cynomolgus monkeys provide the most appropriate in vivo model for evaluating the PK-PD of fusion proteins that bind to PCSK9, because binding to PCSK9 in this model results in a decrease in free PCSK9 and LDL-C; in addition, HSA can be effectively recycled via the NHP FcRn receptor.

[0057] The in vivo binding affinity of fusion proteins that bind PCSK9 to hPCSK9 was studied using hPCSK9 transgenic mice, and the ability to recycle fusion proteins that bind PCSK9 via hFcRn was also assessed in hFcRn mice. The human FcRn mouse model carries a null mutation of the mouse FcRn gene and a transgene that expresses the human FcRn α chain under the control of its native human promoter. Therefore, these mice serve as a model for evaluating the pharmacokinetics of monoclonal antibodies and human serum albumin.

[0058] For anti-PCSK9 antibodies, there are a number of data sets supporting a relationship between inhibition of systemic free PCSK9 levels and lowering of serum LDL-C. Gadkar K et al., A Mechanistic Systems Pharmacology Model for Prediction of LDL Cholesterol Lowering by PCSK9 Antagonism in Human Dyslipidemic Populations , CPT Pharmacometrics Syst. Pharmacol. 2014; 3(11): 1-9; Squizzato A et al., PCSK9 inhibitors for treating dyslipidemia in patients at different cardiovascular risk: a systematic review and meta- analysis. Intern.Emerg.Med.2017;July:e1-11. Figure 3To estimate the human dose of a PCSK9-binding fusion protein (LIB003, SEQ ID NO: 3) required to achieve the desired target LDL-C lowering levels, allometric scaling of the data from the above studies was incorporated into a translational semi-mechanistic model that included the known relationship between PCSK9 and LDL-C. Figure 2 The expected dose-effect relationship between LIB003 and serum LDL-C was constructed using this model.

[0059] TK-PD in nonhuman primates

[0060] The TK-PD behavior of LIB003 was characterized in a single-dose non-GLP dose-ranging (DRF) study following intravenous (IV) and subcutaneous (SC) administration (IV doses of 10, 30, 100, 200 mg / kg; SC dose of 200 mg / kg) and in a GLP toxicity study following repeated administration for 4 weeks (IV dose of 100 mg / kg; SC doses of 30 and 100 mg / kg), 12 weeks (SC doses of 30 and 100 mg / kg), or 26 weeks (SC doses of 30 and 100 mg / kg).

[0061] The TK assay for LIB003 was constructed as a target capture electrochemiluminescent assay with hPCSK9 as the capture reagent and a ruthenium-labeled rabbit anti-HSA polyclonal antibody as the detection reagent. In NHPs, the assay measures total LIB003 (due to a stronger affinity for the capture reagent hPCSK9 compared to NHP PCSK9). Alternatively, the TK assay uses a LIB003-specific mAb as the capture reagent and a ruthenium-labeled rabbit anti-HSA polyclonal antibody as the detection reagent. This assay format also measures total LIB003. The results of these studies indicate that the kinetics of the total binding to PCSK9 fusion protein are approximately linear and dose-proportional over this dose range after intravenous and subcutaneous administration.

[0062] In the DRF study, there were persistent anti-drug antibody (ADA) responses at all dose levels above 10 mg / kg. After a single dose of LIB003, in most cases, the presence of ADA in this study was associated with a rapid loss of LIB003 exposure, and first of all, a loss of target capture (reduction of free PCSK9). Although ADA was detected in most animals after weekly administration of the fusion protein binding to PCSK9 (4-week, 12-week, and 26-week GLP toxicity studies), exposure to the fusion protein binding to PCSK9 was maintained throughout the dosing interval, and the effect of ADA on TK / PD was only evident in two animals (4-week study), one animal (12-week study), and one animal (26-week study).

[0063] In the absence of ADA effects, LIB003 is slowly cleared, and the serum terminal half-life ranges from 8.3-10.4 days (average 9 days) in the DRF study and from 7.6-9.2 days (average 8.3 days) in the recovered animals in the 4-week GLP toxicity study; this is consistent with the behavior of albumin-like molecules in non-human primates. Similar results were observed in 12-week and 26-week GLP toxicity studies. In the DRF study, after a single IV administration, the clearance (CL) ranged from 5.63-7.65 ml / day / kg (average 6.64 ml / day / kg) and the distribution volume (Vz) ranged from 67.6-103.8 mL / kg (average 86.4 mL / kg) in animals that could be measured.

[0064] Total PCSK9 concentrations (both bound and unbound to LIB003) increased slowly and peaked at approximately 7 days after dosing, when total PCSK9 concentrations were less than 10% of circulating LIB003 concentrations. Target capture was maximal at all doses in the DRF study, as measured by free PCSK9 and LDL reduction, indicating that target inhibition was maximal at the lowest dose tested (10 mg / kg). The results indicate that increasing the dose of LIB003 increases the duration of maximal target capture in the absence of ADA.

[0065] Regardless of dose or route of administration, serum LDL was suppressed by approximately 60%, which is consistent with the maximal pharmacodynamic effect at all dose levels. As expected, the loss of LDL suppression and restoration of baseline levels coincides with the loss of maximal PCSK9 capture. Overall, these studies suggest that the maximal pharmacodynamic effect has been achieved and that higher doses do not produce greater inhibition of free PCSK9 or LDL.

[0066] Compared with slow IV push at the same dose level, C max Lower and T max The late onset indicates absorption from the injection site into the systemic circulation. Following subcutaneous administration, the absolute bioavailability of the subcutaneous route was estimated to be approximately 76% in the DRF study and 66.5%-89.7% in the 4-week GLP toxicity study, but both estimates were affected by the presence of ADA and the inability to characterize the total area under the curve (AUC). A combined analysis of these two studies using a population PK model estimated the bioavailability after subcutaneous administration to be 92% and is likely a more reliable estimate because the model generates total AUC estimates for both routes of administration.

[0067] Predicted PK-PD behavior in humans

[0068] Combine data from multiple studies to predict the expected PK-PD behavior of LIB003 in humans. That is, a population 2-compartment PK-PD binding model describing LIB003 exposure and PCSK9 inhibition was constructed from NHP data (DRF studies and 4-week GLP toxicity studies). When these parameters were extended to humans, the in vivo binding affinity for hPCSK9 was derived from PK-PD studies in hPCSK9 transgenic mice. Since only data after subcutaneous administration were available, a one-compartment PK model was sufficient to describe these data; the model was otherwise identical to the model for NHP data. The observed differences in LIB003 binding affinity to NHP PCSK9 and hPCSK9 were consistent in both in vitro and in vivo derived data, and the K from in vivo derived hPCSK9 transgenic mice was used as the model for NHP data. D Values ​​are used for predictions about people.

[0069] Table 2

[0070]

[0071] The clearance of LIB003 in NHP and hFcRn mice was consistent with the expected clearance of HSA in these animals, and therefore, the clearance of LIB003 was predicted to mimic the clearance of HSA in humans (half-life, 19 days; allometric exponent, 0.74). Other parameters were allometrically converted using the expected coefficients for a therapeutic albumin fusion protein of approximately 77 kDa.

[0072] The overall structure of the NHP and human PK-PD binding models describing the capture and inhibition of free PCSK9 is shown in Figure 2 and Figure 3 Quantitative systems pharmacology models have been previously developed to describe the mechanisms of action of statins and anti-PCSK9 antibodies in humans. Gadkar K et al., A Mechanistic Systems Pha r Macology Model for Predictio n of LDL Cholesterol Lowering by PCSK9 Antagonism in Human Dyslipidemic Populations , CPT Pharmacometrics Syst. Pharmacol. 2014; 3(11). The model was used to provide a predicted association between PCSK9 inhibition and LDL-C reduction over time in humans following a single subcutaneous and intravenous administration of LIB003 in a first-in-human study (FIH).

[0073] Example 2: Formulation and stability evaluation

[0074] The performance of various buffers and excipients in the formulation of fusion proteins that bind PCSK9 was evaluated.

[0075] To determine the appropriate buffer and pH to use in the formulation, 18 different buffer and pH conditions using 6 different buffers at 2 mg / mL LIB003 were analyzed by DSC to evaluate thermal stability and by DLS to evaluate aggregate formation. From these experiments, pH below 5 and above 7 were excluded because for these pH values, protein unfolding begins at a temperature (onset temperature (Tonset)) of <50°C.

[0076] The buffer / pH combinations analyzed are shown below in Table 3. Combinations selected for further screening are shown with bold and underlined pH values.

[0077] Table 3: Buffer / pH Screening

[0078]

[0079]

[0080] After confirming the acceptable pH range and appropriate buffer, the excipients were studied in combination with defined buffers and pH. A total of 15 different buffer / excipient combinations were prepared at 2 mg / mL LIB003 and analyzed by DSC to evaluate thermal stability and by DLS to evaluate aggregate formation. Three of the buffer / excipient combinations were excluded due to onset temperatures below 55°C. In addition, DLS data indicated that the use of sucrose, sorbitol, and low concentrations of NaCl resulted in aggregation, so their use was limited in subsequent formulations.

[0081] The buffer / excipient combinations analyzed are shown below in Table 4. Combinations selected for further screening are shown with bold and underlined excipients.

[0082] Table 4: Buffer / Excipient Screening

[0083]

[0084]

[0085] In order to determine the appropriate buffer and excipient combination for maintaining the stability of LIB003 formulated at high concentrations, 12 different formulations identified in Table 4 above were subjected to solubility screening. These formulations were concentrated to target concentrations of 200 mg / mL, 250 mg / mL, 300 mg / mL, and 340 mg / mL. Turbidity was equal in all formulations, and no aggregation was observed when evaluated by SEC-HPLC. Based on these parameters, these formulations are considered to be equivalent.

[0086] In order to evaluate the potential benefits of adding surfactants to the LIB003 candidate formulations, the eight buffer / excipient combinations in Table 4 were evaluated with or without polysorbate 80 (PS80), for a total of 16 formulations at 250 mg / mL LIB003 (Table 5). Since PS80 can effectively prevent aggregation and particle formation, the ability of the candidate formulations to withstand repeated freeze-thaw and agitation stress was evaluated. Based on recovery, turbidity or aggregation tendency (evaluated by DLS and SEC), no differences were observed. Due to the expected benefits of PS80 after long-term storage and product handling, all formulations containing PS80 have made progress.

[0087] Table 5: Buffer / Excipient and Surfactant Screening

[0088]

[0089]

[0090] LIB003 is stable and under normal storage conditions and shear stress, there is no difference between formulations, even at high LIB003 concentrations, indicating that an alternative method is needed to identify LIB003 formulations that can withstand long-term storage conditions. The formulations identified from the buffer / excipient screening (Table 4) at a target concentration of 340 mg / mL were spiked with 0.02% PS80, stored at 2°C-8°C or 50°C for three weeks, and analyzed by SEC-HPLC. Typically, low levels of high molecular weight (HMW) and low molecular weight (LMW) substances were observed after storage at 50°C for 3 weeks. Based on the low percentage of HMW substances in these formulations, the formulations containing citrate were narrowed to excipients arginine and sorbitol. In the histidine formulation, 150 mM NaCl was selected for other experiments based on the highest percentage of the main peak substance. Finally, succinate was no longer considered due to the increased levels of HMW and LMW substances observed in most succinate formulations stored at 50°C relative to the control samples at 2°C-8°C.

[0091] The results of these analyses are shown below in Table 6; preferred formulations are indicated in bold and underlined font.

[0092] Table 6: SEC-HPLC evaluation results of high concentration LIB003 stored at 50°C for 3 weeks.

[0093]

[0094]

[0095] To further explore candidate formulations, LIB003 was formulated at 250 mg / mL with three buffer / excipient combinations plus PS80 at 3 different pHs (Table 7). The samples were stored at 2°C-8°C or 50°C for three weeks to induce degradation, and the concentration / recovery (A280), microparticles (DLS), aggregation (SEC-HPLC), charge distribution (icIEF), clipping (CE-SDS), efficacy, turbidity and thermal stability (DSC) of LIB003 were analyzed. Comparison of charge distribution, aggregation / microparticle formation tendency and relative efficacy after storage at 50°C for 3 weeks demonstrated certain differences between the formulations (Table 8, Table 9, Table 10 and Table 11). CE-SDS curves, recovery, turbidity and thermal stability were comparable in the formulations.

[0096] Table 7. Candidate formulations before storage

[0097]

[0098]

[0099] Table 8. icIEF changes in charge distribution after storage at 50°C for 3 weeks

[0100]

[0101]

[0102] Table 9. SEC-HPLC changes in HMW and LMW materials after storage at 50°C for 3 weeks

[0103]

[0104] Table 10. Potency as a function of storage temperature

[0105]

[0106]

[0107] Given that the goal is to deliver LIB003 in the form of 0.25-1.5 mL SC injection using an automatic syringe with a 27G or smaller caliber needle, additional experiments were performed to determine whether LIB003 formulated at 250 mg / mL has properties suitable for SC injection via an automatic syringe. The viscosity, weight-gram molecular osmotic concentration, and microparticles of the first three formulations with respective center point pH at 250 mg / mL were evaluated. The goal is viscosity <15 cP, weight-gram molecular osmotic concentration of 250-350 mOsm, and microparticle levels far below regulatory limits. The results are shown in Table 11 below. In all formulations, microparticle levels were quite low.

[0108] Table 11: Results of Viscosity and Osmolality Analysis

[0109]

[0110]

[0111] The fusion protein binding to PCSK9 was formulated as a sterile injection solution (subcutaneous) in 20mM histidine, 150mM NaCl, 0.02% (w / v) polysorbate 80, pH 6.8 for further stability studies. These stability studies included evaluations under long-term storage conditions, accelerated conditions, and pressurized conditions. The expected long-term storage temperature of the fusion protein product binding to PCSK9 is 5°C ± 3°C, and the temperature for short-term storage (≤1-6 months) is 25°C ± 3°C.

[0112] Stability data for 1 month were obtained at the expected storage temperature (5°C ± 3°C) as well as accelerated storage conditions (25°C ± 2°C / 60 ± 5% RH) and pressurized storage conditions (40°C ± 2°C / 75 ± 5% RH).

[0113] For the exemplary PCSK9-binding fusion protein product batch, the data for all evaluation parameters in the stability protocol, including stability-indicating parameters (icIEF, potency, CE-SDS, and SEC-HPLC), were within the acceptance criteria at the time point examined (1 month) and under each storage condition. In addition, no significant increase in aggregates or degradation products or decrease in potency was observed under long-term storage conditions. Under accelerated and stressed conditions, a slight increase in aggregates (about 1%) was observed by SEC-HPLC, but these results were well within the main peak acceptance criteria for SEC-HPLC, and no changes in potency were observed. Moreover, under stressed conditions, a slight increase in fragments (about 2%) was observed by reducing CE-SDS, but again no changes in potency were observed. A slight increase in acidic charge variant materials (about 1%-4%) was observed by icIEF under each storage condition, with a concomitant decrease in main peak materials (note that the opposite trend was observed for the drug substance, indicating that the observed changes were within the variability of the method). No changes in appearance, physicochemical parameters, or potency were observed. Taken together, the stability data demonstrate that the PCSK9-binding fusion protein product is stable for 1 month under the evaluated long-term, accelerated, and stressed storage conditions.

[0114] In addition, 18 months of long-term stability data were obtained at the expected storage temperature (5°C ± 3°C), 9 months of long-term stability data were obtained under accelerated storage conditions (25°C ± 2°C / 60±5%Rh), and three months of long-term stability data were obtained under pressurized storage conditions (40°C ± 2°C / 75±5%RH).

[0115] For the exemplary PCSK9-binding fusion protein product batch, data for all evaluation parameters in the stability protocol, including stability-indicating parameters (icIEF, potency, CE-SDS, and SEC-HPLC), were within the acceptance criteria at the time points examined and at each storage condition. In addition, no significant increase in aggregates or degradant materials or decrease in potency was observed under long-term storage conditions. A slight increase in aggregates (about 1%) was observed by SEC-HPLC under all storage conditions, but these results were well within the main peak acceptance criteria for SEC-HPLC, and no changes in potency were observed (within the assay variability). Moreover, a slight increase in fragmentation (up to about 2%) was observed by reducing CE-SDS under long-term storage conditions, but again no corresponding changes in potency were observed, and the changes can be considered to be within the variability of the method. The increase in fragmentation obtained by reducing and non-reducing CE-SDS was associated with increased time and temperature (up to about 5% change under pressurized conditions). Fluctuations in acidic and main peak materials were observed by icIEF under long-term storage conditions (up to about 8%), but the fluctuations were within the variability of the method. The icIEF results under accelerated and stressed conditions initially appeared to indicate a potential positive trend for acidic material (up to approximately 7%) and a corresponding negative trend for the main peak material, but no concomitant changes in potency were observed. However, other time points indicated that the minor changes observed may also be fluctuations related to method variability. Subvisible particles appeared to fluctuate slightly over time but remained within acceptable limits (note that atypical results were observed for particles ≥2μm and ≥5μm at 9 months under both long-term and accelerated conditions; however, particle counts returned to expected levels at the 12-month and 18-month time points). No changes in appearance, physicochemical parameters, or potency were observed.

[0116] Additionally, long-term stability data for 12 months were obtained at the expected storage temperature (5°C ± 3°C), and long-term stability data for 12 months were obtained under accelerated storage conditions (25°C ± 2°C / 60 ± 5% RH).

[0117] For the exemplary PCSK9-binding fusion protein product batch, the data of all evaluation parameters in the stability protocol, including stability indicating parameters (icIEF, potency, CE-SDS and SEC-HPLC), were within the acceptance criteria at the time points examined and under each storage condition. In addition, no significant increase in aggregates or degradation substances or decrease in potency was observed under long-term storage conditions. A slight increase in aggregates (up to about 1%) was observed by SEC-HPLC under both storage conditions, but these results were well within the main peak acceptance criteria of SEC-HPLC, and no changes in potency were observed (within the assay variability). In addition, a slight increase in fragments (up to about 2%) was observed under long-term storage conditions by reducing CE-SDS conditions, a slight increase in fragments (about 3%) was observed under pressurized storage conditions, and a slight increase in fragments (about 3%) was observed under pressurized storage conditions by non-reducing CE-SDS, but similarly no corresponding changes in potency were observed (within the assay variability). An increase in acidic charge variant species (up to approximately 5%) was observed by icIEF under both storage conditions, with a concomitant decrease in the main peak species (this may be attributed to assay variability as later time points had lower percentages of acidic species). No changes in appearance, physicochemical parameters or potency were observed.

[0118] Embodiment 3: First Second-rate Human studies

[0119] LIB003 was studied in a Phase 1 SAD study, which included 63 subjects aged ≥18 years and ≤70 years, 24 women and 39 men, 45 received LIB003 and 18 received placebo, and were monitored for at least 43 days after administration. The study was placebo-controlled and double-blind. There were 7 subjects in each of the 9 groups: 5 patients treated with LIB-003 and 2 patients treated with placebo, so that 43 subjects received LIB003 treatment and 18 received placebo treatment. 25mg, 75mg, 150mg, 300mg and 600mg of LIB003 subcutaneous doses were administered to healthy subjects who were on a stable diet, did not receive lipid-lowering therapy, and had a baseline LDL-C ≥100 and ≤190mg / dL. In addition, 150mg and 300mg doses SC were administered to patients who received stable statin therapy and had a baseline LDL-C ≥100mg / dL. All subjects had TG ≤ 250 mg / dL. Two additional cohorts of healthy subjects not receiving lipid-lowering therapy and with lipid inclusion criteria identical to their SC cohort received LIB003 300 mg and 600 mg IV.

[0120] Summary of Safety Results

[0121] All 63 subjects completed the study, and no patient withdrew or terminated the study before Day 43. Overall, LIB003 was safe and well tolerated following a single SC and IV dose in both healthy subjects and patients with hypercholesterolemia receiving statin therapy.

[0122] Summary of Pharmacodynamic (Efficacy) Results

[0123] The average reduction in free PCSK9 was rapid at all doses and reached more than 99% within 12 hours, and this reduction lasted for at least 3 weeks (Day 22) in almost all subjects in the group receiving ≥150mg LIB003 who were not receiving lipid-lowering therapy. The LIB003 300mg dose maintained 99% free PCSK9 inhibition within 29 days, but in the 150mg dose subjects who were not receiving lipid-lowering therapy, free PCSK9 fell to 12% of baseline and to 54% of baseline in subjects receiving statins. The lower degree of reduction in free PCSK9 was reflected in the reduction of LDL-C and apolipoprotein B, in which subjects who were not receiving lipid-lowering therapy maintained a greater degree of reduction. However, in patients receiving statins, these results did not last within 4 weeks (Day 29). In subjects treated with non-statins and statins, a single 300mg dose can reduce free PCSK9, LDL-C and apolipoprotein B more steadily and to the greatest extent. Furthermore, based on previous data from mAb studies, it is expected that multiple dosing will result in longer duration of free PCSK9 inhibition and LDL-C lowering. Furthermore, extensive previous data show that patients receiving high-intensity statins and patients with FH have higher baseline PCSK9 levels and may have increased PCSK9 synthesis, and thus doses of 300 mg or higher will be required to fully suppress free PCSK9 and LDL-C for 4 weeks.

[0124] Summary of Pharmacokinetic Results

[0125] C of total LIB003 from SC dose max , AUC 0-t and AUC inf There was a dose-proportional increase in total LIB003 between 75 mg and 300 mg, and minimal over-dose proportionality between 25 mg and 75 mg (4-fold to 5-fold increase) and between 300 mg and 600 mg (3-fold increase). Similarly, statin-treated subjects showed a dose-proportional increase in total LIB003 over the 150 mg to 300 mg dose range, but total LIB003 exposure (AUC 0-t and AUC inf) were generally lower than the exposure in subjects treated with non-statin drugs. max , AUC 0-t and AUC inf Increase in proportion to the dose.

[0126] The median T of total LIB003 at all SC dose levels max The range was 72 to 168 hours (range variable: 36 to 220 hours). T-HALF, CL / F, and Vz / F were also similar at the SC doses tested and when LIB003 was administered with a statin. The median T of total LIB003 was max The range was 0.33 to 1.08 hours. T-HALF, CL, and Vz were similar at both IV doses tested.

[0127] The absolute bioavailability of total LIB003 ranged from 67% to 111% following a single SC dose of 300 mg and 600 mg LIB003, respectively.

[0128] Rationale for Phase 2 Dose Levels

[0129] Based on the free PCSK9 and LDL-C data from Phase 1 and the goal of achieving at least Q4W dosing in a volume consistent with a single SC injection using an auto-injector (≤1.5 mL), a Phase 2 dose-finding study is planned in approximately 80 patients with ASCVD or high risk of ASCVD or HeFH without CVD who are receiving stable statins and / or ezetimibe. Doses selected for Q4W dosing in this Phase 2 dose-finding study include 150 mg, 300 mg, and 350 mg. All 3 doses are expected to be safe based on human LIB003 exposures up to 600 mg (SC and IV) in Phase 1 and the lack of findings at levels achieved in a 12-week non-human primate GLP toxicology study.

[0130] Example 4: To evaluate the need want LIB003 to further reduce LDL-C in patients on stable lipid-lowering therapy Randomized, double-blind, placebo-controlled, phase 2 dose-finding study of efficacy and safety

[0131] LIB003 was studied in a 12-week randomized, double-blind, placebo-controlled, dose-finding Phase 2 study, followed by a 4-week follow-up assessment period to assess the percent change from baseline in LDL-C levels at various doses of LIB003 given monthly (Q4W) as calculated by the Friedewald formula at Weeks 10 and 12 and averaged at Week 12. A total of 81 men and women aged ≥18 years with atherosclerotic cardiovascular disease (ASCVD) or at high ASCVD risk (≥10% 5-year or ≥7.5% 10-year risk) and with a calculated LDL-C ≥80 mg / dL for subjects with ASCVD or CVD risk, or ≥100 mg / dL for subjects with heterozygous familial hypercholesterolemia and no CVD were enrolled in the study. All subjects had TG≤400mg / dL under stable lipid-lowering oral drug therapy (such as statins with or without ezetimibe). These subjects were divided into 3 active substance groups and 1 matching placebo treatment group. In each group, three LIB003 subjects were assigned to each placebo subject, i.e., 20 LIB003 subjects and 20 placebo subjects in each treatment group. 150mg, 300mg or 350mg LIB003 or placebo subcutaneous doses were subcutaneously administered to subjects with hypercholesterolemia who received stable diet and oral LDL-C lowering drug therapy every month (Q4W).

[0132] A summary of the demographics of the subjects enrolled in this study is shown in Table 11 below.

[0133] Table 11: Demographics of the Subject Group

[0134]

[0135] Summary of Safety Results

[0136] In general, in this study, LIB003 is safe and generally well tolerated as a subcutaneous dose of up to 350mg Q4W. All doses of LIB003 (150mg, 300mg and 350mg) are well tolerated and do not show any safety issues. Among these 81 subjects, 79 subjects completed the study and 2 subjects withdrew from the study. Withdrawal from the study was not due to adverse events. In addition, no adverse events led to death in this study.

[0137] A summary of treatment-emergent adverse events (TEAEs) experienced by subjects during the study is shown in Table 12 below.

[0138] Table 12: TEAEs of subjects during the study

[0139]

[0140] A total of 43 of 81 subjects (53%) had TEAEs, including 10 of 20 subjects (50%) who received placebo and 33 of 61 subjects (54%) in the LIB003 combination treatment group. Most TEAEs were mild to moderate in severity. The most commonly reported TEAEs were fatigue, injection site bruising, upper respiratory tract infection, and dyspnea. All other TEAEs were reported by ≤3 subjects.

[0141] Only 6 of 81 subjects (7%) had TEAEs related to study drug, including 2 of 20 subjects (10%) receiving placebo and 4 of 61 subjects (7%) in the LIB003 combination group. Most TEAEs related to study drug were mild in severity; none were considered severe. The most frequently reported TEAE related to study drug was injection site erythema. All other TEAEs related to study drug were reported by ≤1 subject.

[0142] Six of 81 subjects (7%) had SAEs, one (5%) in the placebo group, and five (8%) in the LIB003 combination group; none were considered related to study drug. Five SAEs (one (5%) in the placebo group and four (7%) in the LIB003 combination group) were severe in severity, and one (2%) in the LIB003 combination group was moderate in severity.

[0143] There were no TEAEs associated with abnormal laboratory values. There were no clinically significant increases or trends in liver function tests (ALT, AST, or bilirubin) in any treatment group, and there were no differences between the placebo group or the LIB003 treatment group. Specifically, no subject experienced an increase in ALT or AST>3×ULN, and no subject had bilirubin>2×ULN. Most subjects in all treatment groups did not have a sustained increase in CK (which is related to exercise or activity), and none exceeded 5×ULN. There were no clinically significant increases or differences between treatment groups in renal function, glucose, other chemistry or hematology parameters.

[0144] There were no clinically significant findings in vital signs, ECG, and physical examination results. There were a total of five documented injection site erythema events at the 15-minute post-dose time point following study drug administration. One subject in the placebo group also reported injection site pruritus 15 minutes after the first day dose. All injection site reactions occurring 15 minutes after the dose were mild in severity, with 1 in the placebo group and 4 in the LIB003 combination treatment group.

[0145] Summary of Efficacy Results

[0146] All LIB003 doses tested (150 mg, 300 mg and 350 mg) resulted in rapid, sustained and significant average decreases in LDL-C and free PCSK9 levels. The maximum average decrease in LDL-C was observed in the 300 mg LIB003 dose group. Consistent with previous studies, the higher 350 mg LIB003 dose did not further reduce LDL-C. It was found that the 150 mg dose was insufficient to obtain the maximum LDL-C reduction in the entire four weeks between doses.

[0147] A summary of the efficacy data from this study is shown below in Table 13.

[0148] Table 13: Efficacy Data

[0149]

[0150]

[0151] Following dosing in LIB003, LDL-C levels continued to decrease significantly on average from baseline to the mean of Week 10 and Week 12 LOCF and to Week 12 LOCF as calculated by the Friedewald formula. The largest LS mean percentage change difference (95% CI) in LDL-C from baseline to the mean of Week 10 and Week 12 LOCF and to Week 12 LOCF between the LIB003 group and the placebo group occurred in the 300 mg LIB003 dose group and was -76.1% ([-86.0%, -66.2%], p<0.0001) and -77.3% ([-90.5%, -64.1%], p<0.0001), respectively. Similar findings were obtained when the mean percentage change in LDL-C levels was assessed by preparative ultracentrifugation and the Hopkins formula.

[0152] Consistent with previous studies showing that once maximal PCSK9 inhibition is achieved, no further LDL-C reductions occur, the higher dose of 350 mg LIB003 did not result in further reductions in LDL-C levels. Although the 150 mg dose achieved similar reductions as the 300 mg and 350 mg doses after biweekly dosing, it was insufficient to maintain maximal LDL-C reductions across the four weeks between doses.

[0153] Following the LIB003 dose, there was a sustained and substantial mean decrease in free PCSK9 levels from baseline to the mean of Week 10 and Week 12 LOCF and to Week 12 LOCF. The LS mean percentage change differences (95% CI) in free PCSK9 levels from baseline to the mean of Week 10 and Week 12 LOCF and to Week 12 LOCF between the LIB003 group and the placebo group occurred in the 300 and 350 mg groups, which were -89.7% ([-100.0%, -79.4%], p<0.0001) and -92.8% ([-102.9%, -82.6%], p<0.0001), respectively. The LS mean percentage difference (95% CI) in free PCSK9 levels from baseline to Week 12 LOCF between the LIB003 group and the placebo group was -84.1% ([-99.5%, -68.7%], p < 0.0001) and -90.2% ([-105.4%, -75.0%], p < 0.0001) in the 300 and 350 mg groups, respectively. Although the 350 mg LIB0003 dose inhibited slightly more PCSK9 than the 300 mg dose at Week 12 (90.2% vs. 84.1%, respectively), this did not result in greater LDL-C efficacy.

[0154] In addition, after LIB003 dosing, total PCSK9 levels continued to decrease significantly from baseline to the mean of LOCF at Week 10 and Week 12, and to Week 12 LOCF. For LIB003 150 mg, 300 mg, and 350 mg groups, the mean percentage change from baseline to the mean of LOCF at Week 10 and Week 12 was 95.555%, 90.273%, and 90.080%, respectively. For LIB003 150 mg, 300 mg, and 350 mg groups, the mean percentage change from baseline to Week 12 LOCF was 75.165%, 78.188%, and 86.811%, respectively. In contrast, placebo showed the smallest mean percentage change (≤3.044%) at the same time point.

[0155] In addition, non-HDL-C levels continued to have large mean decreases from baseline to the mean of Week 10 and Week 12 LOCF and to Week 12 LOCF, and TC levels continued to have moderate mean decreases. However, there were only minimal mean changes in HDL-C levels from baseline to the mean of Week 10 and Week 12 LOCF and to Week 12 in both the LIB003 cohort and the placebo group. In addition, the percentage changes in VLDL-C and TG levels between each LIB003 cohort and the placebo group were compared using ANOVA models, showing small mean decreases in VLDL-C and TG levels from baseline to the mean of Week 10 and Week 12 LOCF and to Week 12 LOCF.

[0156] Furthermore, following the LIB003 dose, apolipoprotein B levels continued to decrease significantly on average from baseline to LOCF Week 12. The largest LS mean percentage change difference (95% CI) in apolipoprotein B between the LIB003 group and the placebo group occurred in the 300 mg group and was -58.4% ([-68.9%, -48.0%], p<0.0001).

[0157] In addition, Lp(a) levels continued to decline moderately from baseline to LOCF at week 12 after all LIB003 doses. The maximum LS mean percentage change difference (95% CI) in Lp(a) between the LIB003 group and the placebo group occurred in the 300 mg group and was -28.7% ([-42.6%, -14.8%], p < 0.0001). This reduction is consistent with the reduction achieved by PCSK9 monoclonal antibodies at equivalent doses and dosing frequencies, which inhibited free PCSK9 and LDL-C similarly to LIB003 300 mg Q4W.

[0158] Apolipoprotein A1 levels showed minimal mean changes from baseline to Week 12 LOCF for both the LIB003 population and the placebo group.

[0159] Based on the data from the above Phase 2 study, the LIB003 300 mg subcutaneous Q4W dose was selected for open-label extension and Phase 3 studies.

[0160] sequence

[0161] PCSK9 binding motif

[0162]

[0163] HSA amino acid sequence

[0164]

[0165] Fusion protein binding to PCSK9

[0166]

[0167]

Claims

1. A composition comprising one or more unit doses of a fusion protein that binds to PCSK9 having the amino acid sequence of SEQ ID NO: 3, wherein the unit dose comprises 250 mg to 350 mg of the fusion protein that binds to PCSK9 at a concentration of at least 200 mg / mL; and a pharmaceutically acceptable carrier.

2. The composition of claim 1, comprising at least 250 mg / mL of the PCSK9-binding fusion protein.

3. The composition of claim 1, comprising at least 300 mg / mL of the PCSK9-binding fusion protein.

4. The composition of claim 1, comprising a unit dose of 275 mg to 325 mg of the PCSK9-binding fusion protein.

5. The composition of claim 1, comprising a unit dose of 300 mg of the PCSK9-binding fusion protein.

6. The composition of any one of claims 1 to 5, wherein the pharmaceutically acceptable carrier comprises a buffer, an isotonic agent and a solvent.

7. The composition of claim 6, wherein the buffer comprises an amino acid buffer, a citrate buffer, a phosphate buffer, an acetate buffer, a succinate buffer and / or a bicarbonate buffer.

8. The composition of claim 7, wherein the buffer comprises L-histidine / L-histidine monohydrochloride.

9. The composition of claim 8, wherein the L-histidine / L-histidine monohydrochloride is present at 1 mg / mL to 10 mg / mL and the pH of the composition is maintained at 6.

8.

10. The composition of claim 6, wherein the isotonic agent comprises one or more of sodium chloride, dextrose, sucrose, glycerol, mannitol, sorbitol, arginine, or potassium chloride.

11. The composition of claim 10, wherein the isotonic agent comprises sodium chloride.

12. The composition of claim 11, wherein the sodium chloride is present at 2 mg / mL to 20 mg / mL.

13. The composition of claim 6, wherein the pharmaceutically acceptable carrier comprises a surfactant.

14. The composition of claim 13, wherein the surfactant is a polysorbate surfactant.

15. The composition of claim 14, wherein the surfactant is polysorbate-80.

16. The composition of claim 6, comprising 20 mM histidine buffer, 150 mM NaCl, 0.02% (w / v) polysorbate-80, and the pH of the composition is 6.

8.

17. The composition of claim 6, wherein the composition does not contain a surfactant.

18. The composition of claim 6, wherein the solvent is water.

19. The composition of any one of claims 1 to 5, further comprising a preservative.

20. The composition of claim 19, wherein the preservative is selected from phenol, m-cresol and sodium benzoate.

21. The composition of any one of claims 1 to 5, wherein the composition is contained in an injection pen.

22. The composition of claim 21, wherein the injection pen contains and delivers a unit dose of 275 mg to 300 mg of the PCSK9-binding fusion protein.

23. The composition of claim 22, wherein the injection pen contains and delivers a unit dose of 300 mg of the PCSK9-binding fusion protein.

24. The composition of claim 21, wherein the volume of the one or more unit doses does not exceed 1.5 mL.

25. The composition of claim 21, wherein the volume of the one or more unit doses does not exceed 1 mL.

26. The composition of claim 24, wherein the volume of the one or more unit doses does not exceed 0.8 mL.

27. Use of a composition as claimed in any one of claims 1 to 26 in the preparation of a medicament for treating hypercholesterolemia, atherosclerotic cardiovascular disease or a subject at high risk of atherosclerotic cardiovascular disease in a subject.

28. The use of claim 27, wherein the subject suffers from familial hypercholesterolemia.

29. The use of claim 27 or 28, wherein the subject is not undergoing statin therapy, or is not undergoing treatment with lipid-lowering oral therapy.

30. The use of claim 27 or 28, wherein the subject is not undergoing statin therapy or another lipid-lowering oral therapy.

31. The use of claim 29, wherein the subject has statin intolerance.

32. The use of claim 27 or 28, wherein the composition is administered subcutaneously, intramuscularly, intradermally or intravenously.

33. The use of claim 27 or 28, wherein the subject receives a unit dose of the composition from once a week to once every two months.

34. The method of claim 33, wherein the subject receives a unit dose of the composition once a month or once every 4 weeks, wherein the unit dose of the composition is administered subcutaneously.

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